Nucleic acid base pairing is an extremely high affinity and specific interaction. For this reason, nucleic acid hybridization assays have been devised for a variety of diagnostic purposes.
Under laboratory conditions, hybridization assays can be extraordinarily sensitive, detecting femtogram amounts of a specific molecule. However, several technical limitations have prevented widespread use of hybridization analysis in commercial diagnostic techniques.
First, use of high activity hybridization probes requires stringent procedures for separating unhybridized (or improperly hybridized) and hybridized probe. This separation can be facilitated by the use of solid phase hybridization formats, in which either the sample nucleic acid or the probe that is complementary to the desired target is immobilized on a solid support. In the latter strategy, the immobilized probe, hereafter referred to as the “capture” probe, is usually unlabeled, and the hybridization is detected by a second hybridization probe that binds the sample at a position separate from that recognized by the capture probe. Hybridized and unhybridized species can be separated by washing the support.
A second limitation of hybridization assays is that efficient hybridization of samples containing low concentrations of target nucleic acids frequently requires lengthy incubations (up to several hours) under carefully controlled conditions. Unfortunately, use of solid phase assays exacerbates this problem, since immobilized nucleic acids virtually always hybridize with slower kinetics than nonimmobilized ones.
For these reasons, a number of workers have sought methods to perform solid phase hybridizations with better kinetics and efficiency. Several groups have found that inclusion of high molecular weight polymers such as dextran sulfate or polyethylene glycol improves solid phase assay performance, albeit modestly. (Wieder and Wetmur, Biopolymers, 20:1537 (1981); Wetmur, Biopolymers, 14:2517 (1975); Yokota and Oishi, Proc. Natl. Acad. Sci. USA, 87:6398 (1990)). Several groups have developed chromatographic solid phase hybridization methods that show improvements. In general, it has been found that flowing the solution phase nucleic acid strand over (or through) the solid support bearing the immobilized strand improves both kinetics and efficiency of hybridization. MacMahon and Gordon, U.S. Pat. No. 5,310,650, describes immobilized target molecules on nitrocellulose filters, with labeled probe flowing through the immobilized target regions by capillary action. In a similar experiment, Reinhartz et al., Gene, 136:221-226 (1993)) immobilized capture probes on paper filters and flowed labeled single-stranded PCR products through the capture probe region, again using capillary action. Others have demonstrated improved hybridization assays by passing samples through an HPLC column containing silica particles covalently modified with capture probes. (Tsurui et al., Gene, 88:233-239 (1990)).
However, despite these advances, there remains a need for a hybridization analysis method that is not only accurate, but fast, efficient and simple to use.